WO1998058079A1 - Diagnostic par biocapteurs a acides nucleiques - Google Patents

Diagnostic par biocapteurs a acides nucleiques Download PDF

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Publication number
WO1998058079A1
WO1998058079A1 PCT/CA1998/000402 CA9800402W WO9858079A1 WO 1998058079 A1 WO1998058079 A1 WO 1998058079A1 CA 9800402 W CA9800402 W CA 9800402W WO 9858079 A1 WO9858079 A1 WO 9858079A1
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Prior art keywords
nucleic acid
biosensor
fiber
immobilized
nucleic acids
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PCT/CA1998/000402
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English (en)
Inventor
Ulrich J. Krull
Paul A. Piunno
Robert H. E. Hudson
Masad Damha
Andre H. Uddin
Original Assignee
Krull Ulrich J
Piunno Paul A
Hudson Robert H E
Masad Damha
Uddin Andre H
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Priority claimed from CA002208165A external-priority patent/CA2208165A1/fr
Application filed by Krull Ulrich J, Piunno Paul A, Hudson Robert H E, Masad Damha, Uddin Andre H filed Critical Krull Ulrich J
Priority to AU70244/98A priority Critical patent/AU755913B2/en
Priority to JP50343899A priority patent/JP2002511934A/ja
Priority to EP98916750A priority patent/EP0991777A1/fr
Priority to US09/446,222 priority patent/US6503711B1/en
Priority to NZ502303A priority patent/NZ502303A/en
Priority to CA002294603A priority patent/CA2294603A1/fr
Publication of WO1998058079A1 publication Critical patent/WO1998058079A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/902—Specified use of nanostructure
    • Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/924—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis using nanostructure as support of dna analysis

Definitions

  • the present invention is directed generally to biosensors that are useful in the identification and analysis of biologically significant nucleic acids.
  • the biosensors of the present invention and their applied methods provide a means for the direct analysis of nucleic acid hybridization and, therefore, have application to a myriad of biological fields including clinical diagnostics.
  • the detection and identification of microorganisms is a problem common to many areas of human and veterinary health.
  • pathogenic species such as Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli, which are causative agents of major food borne epidemics
  • detection and identification of infectious diseases caused by pathogenic microorganisms and viruses is a first step in diagnosis and treatment.
  • RNAs are difficult to assay by PCR but are very important for human viral detection. In general, PCR needs to be automated for acceptance as a practical diagnostic tool. Hybridization methods require as much as three or four days to complete results. Although the actual hybridization step can be as short as 18 hours, the entire detection process of a DNA DNA hybrid can take as long as three days with a radioisotope marker.
  • Biosensors developed to date begin to overcome drawbacks associated with the current state of the art in detecting and identifying microorganisms.
  • a biosensor is a device which consists of a biologically active material connected to a transducer that converts a selective biochemical reaction into a measurable analytical signal (Thompson et al., 1984. Trends in Analytical Chemistry, 3: 173; Guilbault, 1991 , Current Opinion in Biotechnology, 2: 3).
  • the advantages offered by biosensors over other forms of analysis include the ease of use (by non-expert personnel), low cost, ease of fabrication, small size, ruggedness, facile interfacing with computers, low detection limits, high sensitivity, high selectivity, rapid response, and reusability of the devices.
  • Biosensors have been used to selectively detect cells, viruses, other biologically significant materials, biochemical reactions and immunological reactions by using detection strategies that involve immobilization of enzymes, antibodies or other selective proteins onto solid substrates such as quartz and fused silica (for piezoelectric and optical sensors) or metal (for electrochemical sensors) (Andrade et aL, 1990, Biosensor Technology: Fundamentals and Applications, R. P. Buck, W. E. Hatfield, M. Umana, E. F. Bowden, Eds., Marcel Dekker Inc., NY, pp. 219; Wise, 1990, Bioinstrumentation: Research, Developments and Applications, Butterworth Publishers, Stoneham, MA).
  • quartz and fused silica for piezoelectric and optical sensors
  • metal for electrochemical sensors
  • a chemical denaturation scheme was observed to be the preferred embodiment for sensor regeneration as exposure of the oligonucleotide functionalized optical sensor to temperatures exceeding 52°C caused irreversible damage to the device, owing to denaturation of the avidin used for immobilization.
  • This limitation renders the device function labile against sterilization techniques, such as autoclaving, and also indicates that rigorous cleaning of the sensor surface, such as by sonication, would also compromise the integrity of the sensor via denaturation of the affinity pair used to anchor the probe oligonucleotide.
  • a competitive binding assay was employed by Abel and co- workers.
  • Detection of the unlabelled analyte was done by pre-treatment of the sensor with fluorescein labeled "tracer-DNA” followed by monitoring decreases in the fluorescence intensity of the sensor upon exposure to and subsequent displacement of the tracer-DNA by complement analyte nucleic acid.
  • the dose- response curves reported by Abel et al. show a detection limit of 132 pmol (8 x 10 13 molecules) for this detection strategy.
  • this device cannot be classified as a biosensor technology due to the necessity for external treatment with tracer-DNA in order to achieve transduction.
  • Refractive index alterations affect the penetration depth of the evanescent wave emitted from the first waveguide into which optical radiation is launched. This standing wave of electromagnetic radiation subsequently propagates into (and thus transfers optical radiation to) the second waveguide. Therefore, the device is sensitive to refractive index alterations occurring within a volume surrounding the first waveguide with a thickness of ca. one wavelength of the light propagating within that waveguide.
  • One of the arms of the waveguide may be functionalized with immobilized nucleic acid molecules which serves to provide selective binding moieties.
  • the change in refractive index of the thin film of nucleic acids on the first waveguide upon the occurrence of hybridization with target nucleic acid sequences alters the quantity of light transferred to the second waveguide, thereby providing a means of signal transduction.
  • Hybridization events may then be identified based on changes in the output ratios of the two waveguide arms in the coupled fiber system.
  • One limitation of this technology lies in the fact that any alterations in refractive index near the surface of the waveguides will provide alterations in the output ratios of the two fibers. Therefore, non-specific binding events (such as protein adsorption) will provide false positive results.
  • the target nucleic acid is functionalized with a fluorescently detectable agent (by chemical or enzymatic methods) as a first step prior to detection.
  • a fluorescently detectable agent by chemical or enzymatic methods
  • the fluorescent agent is then bound in close proximity to the waveguide surface where it may be excited by evanescent wave formation and emission from the fluorophore collected and quantitatively measured.
  • hybridization between the immobilized oligonucleotide and the target sequence is first done.
  • a fluorescently labeled oligonucleotide present in the system may then undergo hybridization with all or a portion of the remainder of the target sequence not hybridized with the immobilized sequence.
  • the binding of the third (labeled) oligonucleotide provides a fluorescent species bound in close proximity to the waveguide which may furnish transduction via evanescent excitation and collection of the emitted radiation.
  • Abel et al. a method for the detection of nucleic acids not pre-labeled with a fluorescent moiety via a competitive binding assay is described.
  • Detection of the unlabelled analyte was done by first pre-treating the optical sensor with immobilized probe nucleic acid with fluorescein labeled "tracer-DNA".
  • the quantity of tracer-DNA may be monitored via the evanescent excitation and collection motif. Binding of the analyte could be followed by monitoring decreases in the fluorescence intensity from the sensor as a function of the displacement of the tracer-DNA via competitive binding with non-fluorescent analyte nucleic acid in a dose-response convention.
  • fluorescent intercalating dyes e.g. ethidium bromide
  • DNA oligonucleotide arrays have been fabricated using high-resolution photolithography in combination with solid-phase oligonucleotide synthesis.
  • This form of DNA chip technology may be used for parallel DNA hybridization analysis, directly yielding sequence information from genomic DNA segments.
  • the nucleic acid targets Prior to sequence identification, the nucleic acid targets must be fluorescently labeled, either prior to or after hybridization to the oligonucleotide array, via direct chemical modification of the target strand or by use of an intercalant dye subsequent to hybridization on the DNA chip.
  • the hybridization pattern as determined by fluorescence microscopy, is then deconvolved by appropriate chemometric processing to reveal the sequence of the target nucleic acid.
  • the critical angle ( ⁇ c ) for the waveguide/solution interface (0 C WS ) is larger than ⁇ c for the waveguide/biological film interface (0 C WB ), only the evanescent component of the propagated radiation will enter the biological film.
  • the present invention concerns biosensors for direct detection of nucleic acids and nucleic acid analogs.
  • the device comprises a light source, a detector, and an optical element for receiving light from the source and conveying it to an interaction surface of the optical element.
  • a nucleic acid or nucleic acid analog for a particular nucleic acid sequence or structure i.e. which is complementary to the target nucleic acid(s)
  • Fluorescent ligands are provided that will bind into or onto the hybridized nucleic acid complex and fluoresce when stimulated by the light source.
  • An interaction surface is defined to mean a surface of the optical element on which nucleic acid is immobilized, and at which the fluorescent molecules interact with the light.
  • This invention provides biosensors in which the interaction surface is functionalized with nucleic acid probe sequences such that the index of refraction of the immobilized layer (Substrate Linker / Nucleic Acid / Fluorescent Ligand) is equal to or greater than the refractive index at the surface of the waveguide such that the organic coating becomes an extension of the waveguide.
  • the index of refraction of the immobilized layer is dependent, at least in part, on the loading of immobilized molecules and linkers on the surface and the chemical nature of the immobilized molecules and any linkers.
  • Preferred biosensors which offer high-sensitivity and low-detection limits may be realized by activating the interaction surface of an optical element with substrate linker molecules of at least about 25A (Angstrom) in length followed by attachment of a selected probe nucleic acid sequence to that linker.
  • a probe nucleic acid is, at least in part, complementary to a target nucleic acid.
  • the preferred method for attachment of the probe nucleic acid to the substrate linker is by in-situ synthesis of the nucleic acid sequence onto the linker terminus using solid-phase nucleic acid synthesis methods or routine modifications of thereof. Such methods of in-situ synthesis are particularly useful for immobilization of nucleic acids of 50 or fewer bases and more particularly useful for nucleic acids of 30 or fewer bases.
  • the fluorophore may be tethered to the immobilized DNA, for example, by use of a hydrocarbon tether.
  • the use of tethered probes can significantly reduce biosensor response time as the response mechanism is not diffusionally controlled.
  • the associated fluorophore provides for internal calibration of optical source intensity and detector drift. It also provides for calibration of photobleaching, and provides for internal calibration by monitoring bound against free dye by use of, for example, time-resolved fluorescence measurements.
  • the optical element preferably comprises an optical waveguide which also conveys the fluorescent light to the detector.
  • the optical waveguide preferably conveys the emitted light by total internal reflection to the detector.
  • the optical waveguide can comprise an optical fiber, a channel waveguide, or a substrate that confines light by total internal reflection.
  • the fluorescent molecules preferably provide sufficient Stokes shift such that the wavelength of the light source and the wavelength of the fluorescent light are easily separated.
  • the fluorescent molecules can be provided in a solution in which the optical element is immersed, or by a tether to the nucleic acid that is immobilized to the linker.
  • the light source can be any suitable source such as a gas laser, solid state laser, semiconductor laser, a light emitting diode, or white light source.
  • the detector can be any suitable detector such as a photomultiplier tube, an avalanche photodiode, an image intensifier, multi-channel plate, or semiconductor detector.
  • the biosensor system can be a multi- wavelength, multi-fluorescent system.
  • the light coupling of the system can also be modified to allow a multitude of disposable biosensors to be analyzed either sequentially or in parallel.
  • the biosensor system of the present invention can be constructed and used to detect each of a mixture of target nucleic acids (for example, Chlamydia and Gonorrhea in urogenital infections or E.
  • coli and Salmonella during food processing This may be done by using a plurality of fluorophores (which, for example, fluoresce at different wavelengths), each of which is tethered to an immobilized nucleic acid probe that is characteristic of or specific for detection of a given species or strain.
  • fluorophores which, for example, fluoresce at different wavelengths
  • the observed wavelength(s) of fluorescence emission will then be specific for hybridization of a given target nucleic acid to its complementary immobilized probe.
  • the biosensors of the present invention have an improved detection limit and sensitivity with respect to the prior art and are shown to be stable over prolonged storage and severe washing and sterilization conditions. Sensors stored over 1 year in vacuo, in 1 :1 ethanol/water solutions, absolute ethanol, or dry at -20°C provide identical response characteristics to those freshly prepared. Adsorbed fluorescent contaminants accumulated through storage can be removed (as confirmed through fluorescence microscopy investigations) by sonicating the biosensors in 1 :1 ethanol/water where the sensitivity of the device has consistently been observed to increase by a factor of c.a. 2.5 from this pre- treatment with respect to that of freshly prepared biosensors not cleaned before use. Unlike those of the prior art (e.g.
  • the optical biosensors of the present invention have also shown to be thermally stable wherein device function is maintained after sterilization by autoclaving (20 minutes, 120°C, 4 atmospheres over-pressure).
  • the ability to clean and sterilize a biosensor device so that it may be usable in an on-line configuration and/or in clinical applications is a significant advantage yet realized only by the technology reported herein.
  • Biosensors of this invention also allow for more rapid sample analysis with improved response time for signal generation.
  • the present invention also provides a recyclable or disposable biosensor for detecting a target nucleic acid, which biosensor includes an optical element for receiving and conveying light to an interaction surface of the optical element and nucleic acid, for a particular nucleic acid sequence which is complementary to the target nucleic acid, immobilized onto the interaction surface of the optical element.
  • the recyclable or disposable biosensor preferably comprises an optical waveguide, which preferably conveys the light by total internal reflection to the interaction surface of the optical waveguide when the organic coating is of equal or higher refractive index in comparison to the surface of the waveguide.
  • the optical waveguide preferably comprises an optical fiber.
  • Fluorescent molecules are provided in a solution in which the recyclable or disposable biosensor is immersed that will bind upon hybridization of the immobilized nucleic acid with complementary target nucleic acid and fluoresce when stimulated by light.
  • the fluorescent molecules are provided bound by a tether to the immobilized nucleic acid.
  • the present invention provides biosensors for direct analysis of nucleic acid hybridization by use of an optical substrate such as an optical wafer or an optical fiber, and nucleic acids or nucleic acid analogs which have been immobilized onto the optical substrate.
  • Generation of a fluorescence signal upon hybridization to complementary nucleic acids and nucleic acid analogs in a sample may be achieved in a number of different ways.
  • Biosensors of this invention are sufficiently sensitive to directly detect very small quantities of target nucleic acids in a sample without the need to employ nucleic acid amplification methods such as PCR techniques.
  • Biosensors of this invention can have detection limits for target nucleic acids below 10 6 molecules.
  • the optical biosensor comprises nucleic acid strands or nucleic acid analogs of a specific selected sequence immobilized onto activated optical supports.
  • the selected immobilized sequences are capable of binding to target sequences, including sequences characteristic of and selective for viruses, bacteria, or other microorganisms as well as of genetic disorders or other conditions.
  • Biosensors having such characteristic or selective immobilized sequences are useful for the rapid screening of genetic disorders, viruses, pathogenic bacteria and in biotechnology applications such as the monitoring of cell cultures and gene expression.
  • One important avenue which has been widely ignored by the nucleic acid biosensor community is the investigation of multi- stranded (> 3) nucleic acid formation.
  • triple-helical oligonucleotides have been reported to offer potential use as: sequence-specific artificial nucleases ( ⁇ a ⁇ Moser, H.E.; Dervan, P.B. Science, 1987, 238, 645. ⁇ b ⁇ Strobel, S.A.; Doucettestamm, L.A.; Riba, L.; Housman, D.E.; Dervan, P.B. Science, 1991 , 254, 1639.), DNA-binding protein modulators/gene expression regulators ( ⁇ a ⁇ Cooney, M.; Czernuszewicz, Postel, E.H.; Flint, S.J.; Hogan, M.E. Science, 1988, 241, 456.
  • the present invention can also be used to detect the formation of multi-stranded nucleic acid hybrids (for example, formation triple-helical nucleic acids), and therefore could, for example, operate to monitor the effectiveness, dose dependence and intracellular concentration of nucleic acid pharmaceuticals used in gene therapy applications or as an assay to identify multi-strand formation associated with any of the aforementioned potential applications associated with triple-helical oligonucleotides.
  • multi-stranded nucleic acid hybrids for example, formation triple-helical nucleic acids
  • the invention is a biosensor system for detecting a target nucleic acid, which consists of at least three layers, two of which are a waveguide, wherein one layer includes a nucleic acid or nucleic acid analog capable of hybridizing to the target nucleic acid, and wherein a fluorophore is tethered to the nucleic acid or nucleic acid analog and wherein the biosensor functions according to direct excitation.
  • the invention also relates to a biosensor for detecting a target nucleic acid, which comprises an inner layer, a middle layer and an outer layer, wherein
  • the middle layer includes a nucleic acid or nucleic acid analog capable of hybridizing to the target nucleic acid and has refractive index n 2 , which is greater than or equal to refractive index n and
  • the outer layer has refractive index n 3 , which is less than refractive index n 2 . and wherein a fluorophore is tethered to the nucleic acid or nucleic acid analog of the middle layer and wherein the biosensor functions according to direct excitation.
  • the inner layer is an optical fiber or optical wafer and the outer layer is an ambient.
  • the outer layer is an aqueous based solution.
  • the biosensor is useful for detection of triplex formation or multi- stranded nucleic acid formation.
  • the triplex formation preferably involves a branched antisense nucleic acid which inhibits expression of a target nucleic acid sequence by triplex formation with the sequence.
  • the biosensor is useful for detection of nucleic acids of bacteria, viruses, fungi, unicellular or muiticellular organisms or for the screening of nucleic acids of cells, cellular homogenates, tissues or organs.
  • a fluorophore is tethered to a nucleic acid or nucleic acid analog which is one of the layers of a biosensor having at least three layers and the biosensor functions according to direct excitation.
  • the invention also includes the use of a fluorophore for detecting a target nucleic acid.
  • the invention also relates to a method of detecting a target nucleic acid, comprising:
  • Figure 1(b) Synthetic scheme of Brennan et al. used to create alkylamine substrate linker molecules on hydroxylated fused silica surfaces.
  • Figure 1(c) Synthetic scheme of Maskos and Southern used to functionalize hydroxylated fused silica surfaces with GOPS followed by extension with HEG.
  • Figure 1(e) Synthetic scheme used to extend GOPS functionalized substrates with DMT-HEG via a base catalyzed mechanism.
  • Figure 1(f) Synthetic scheme used to covalently link DMT-HEG onto hydroxylated fused silica surfaces via activation with methanesulfonyl chloride.
  • Figure 2 The phenoxyacetyl protecting group used for exocyclic amine (R) protection on nucleoside phosphoramidite synthons.
  • Figure 3(a) Synthetic scheme used to create a hydrocarbon-tethered analogue of Ethidium Bromide.
  • FIG. 3(b) Synthetic scheme used to create a polyether-tethered phosphoramidite analogue of Ethidium Bromide.
  • FIG. 3(b) Synthetic scheme used to create a polyether-tethered analogue of the bis-intercalative fluorescent probe YOYO-1. Removal of the DMT protecting group followed by treatment with ⁇ -cyanoethyl-N,N-diisopropyl phosphityl chloride will yield the tethered YOYO-1 phosphoramidite synthon.
  • Figure 4(a) Schematic diagram of one embodiment of an apparatus used to measure fluorescence intensity from optical fibers coated with immobilized DNA.
  • Figure 4(b) Schematic diagram an example of a dedicated instrument for analysis of nucleic acid samples by the fiber-optic nucleic acid biosensor of the present invention.
  • Figure 4(c) Schematic representation of a biosensor system in which light from a suitable source is directed through a dichroic mirror beam splitter and focused onto a fiber or waveguide coupler and then into an optical fiber having single- stranded nucleic acid bound to the surface thereof, and in which any resultant fluorescent light travels back through the coupler, and passes through the beam splitter and is directed to a photomultiplier detector.
  • Figure 5 Illustration of the operating principles of the fiber-optic nucleic acid biosensor. Hybridization of complement single-stranded oligonucleotide from solution with immobilized nucleic acid probe on biosensor is followed by intercalation of the tethered fluorescent ligand which provides transduction of the selective binding process into a measurable analytical signal.
  • Figure 6. Fluorescent intensity as a function of temperature for the mixed base sequence icosanucleotide functionalized fibers. Upper Curve: response of the optical sensor to 20pmol of linear complement icosanucleotide in the presence of 2.5 x 10 "8 M ethidium bromide. Lower Curve: response of the optical sensor to 2.5 x 10 8 M ethidium bromide.
  • Figure 9(a) Response characteristics of an optical biosensor to complement and non-complement DNA.
  • Figure 9(b) Response characteristics of an optical biosensor to 570 ng ml '1 of complement RNA.
  • Figure 11 Response of a DNA optical biosensor (a) after storage for one month used without cleaning and (b) after storage for eleven months and cleaned by sonication in ethanol for 10 minutes. Note: A 1 -month-old sensor which had been cleaned by sonication (data not shown) provided a response similar to (b).
  • Figure 12. Thermal denaturation profiles of aqueous dA 20 + dT 20 and immobilized dT 20 with aqueous dA 20 .
  • Figure 13 Response of the optical sensor with immobilized nucleic acid probe for Candida albicans to complement DNA.
  • Figure 14 Response of a reagentless biosensor as described in Example 14.
  • the graph measures fluorescence from the tethered dye on the terminus of the immobilized nucleic acid as a function of time after exposure to a sample of 720 ng of cDNA.
  • Figure 15 The structures of dT 10 and compound ⁇ , a branched oligonucleotide with identical oligo(thymidine) chains linked to the 2'- and 3'-positions of a ribose branch-point nucleoside i.e., rA ⁇ 5' : ⁇ , 1 " binds to dA 10 to yield a triple-stranded complex containing only T*AT (reverse Hoogsteen Watson/Crick) base triplets.
  • T*AT reverse Hoogsteen Watson/Crick
  • Figure 16(a) Response ( • ) of the optical sensor with a 5'-end terminated recognition sequence to 40 pmol of linear dT 10 in the presence of 2.5 x 10 "8 M ethidium bromide.
  • Response (X) of the optical sensor to 2.5 x 10 "8 M ethidium bromide and no dT 10 .
  • Figure 16(b) Response ( • ) of the optical sensor with a 3'-end terminated recognition sequence to 40 pmol of linear dT 10 in the presence of 2.5 x 10 8 M ethidium bromide.
  • FIG. 17(a) Photograph of a UV-shadowed native polyacrylamide gel containing single strands, duplex and triple helical complexes of branched and linear controls. DNA samples were loaded in 50mM MgCI 2 , and 30% sucrose. Lanes 4-10 are dT 10 , dT 10 :dA 10 (1 :1),. dT 10 :dA 10 (2.5:1), dT 10 :dA 10 (4:1), dA 10 , I + dA 10 , and i, respectively. As can be noted the dT 10 :dA 10 triplex (lane 7) showed a greater retardation in the mobility relative to the corresponding duplex (lanes 5 and 6). The slowest mobility was observed in lane 9 for I:dA 10 . Note: See Fig. 15 for the structure of ⁇ .
  • FIG 17(b) Photograph of an ethidium bromide stained native polyacrylamide gel (same gel as Figure 17 ⁇ a ⁇ ) containing single strands, duplex and triple helical complexes of branched and linear controls. DNA samples were loaded in 50mM MgCI 2 , and 30% sucrose. Lanes 4-10 are dT 10 , dT 10 :dA 10 (1 :1), dT 10 :dA 10 (2.5:1), dT 10 :dA 10 (4:1), dA 10 , i + dA 10 , and I, respectively. As can be noted the dT 10 :dA 10 triplex (lane 7) showed a slight retardation in the mobility relative to the corresponding duplex (lanes 5 and 6). The slowest mobility was observed in lane 9 for I:dA 10 . Notice that only the duplexes and triplexes showed ethidium bromide fluorescence. Note: See Fig. 15 for the structure of I.
  • Figure 18 Schematic diagram illustrating the experimental concept for light scattering investigations of a two-layer system with t? Fused shunt ⁇ ca > n F , ⁇ m .
  • Figure 19 Schematic diagram illustrating the experimental concept for light scattering investigations of a three-layer system with n Fused Slllca > n F ⁇ m > n Amb ⁇ ent .
  • Figure 20 Schematic diagram illustrating the experimental concept for light scattering investigations of a three-layer system with t7 Fused Slllca ⁇ n Fllm > n Amb ⁇ eni .
  • Figure 21 Schematic diagram of the instrument used for investigations of angularly dependent light scatter.
  • Figure 24 Results of the light scattering experiments done with substrates coated with covalently immobilized oligonucleotides.
  • the invention relates to a biosensor which functions according to an intrinsic mode of operation.
  • chemistries as disclosed in this patent application for attaching linker molecules onto optical waveguide supports (preferably optical fibers) and an automated DNA synthesizer, control over the orientation and a wide range of oligonucleotide packing densities on the waveguide is afforded.
  • immobilized films of oligonucleotides of desired refractive index may be constructed on waveguide supports so that the oligonucleotide film is made to be an extension of the waveguide.
  • the biorecognition element may be a biological material capable of participating in highly selective binding to a target, usually a biologically significant molecule.
  • the transduction element converts the selective binding reaction into a measurable analytical signal.
  • the transduction strategy of Gerdt is too non- selective for the technology to be classified as a biosensor whereas the devices of Fodor, Squirrell, Abel et al., Sutherland et al. and Hirschfeld do not contain a transduction element at all.
  • the extra shortcoming that all intercalant dyes are known or suspected mutagens In addition to the requirement for external reagent treatment, in the cases of Fodor, Sutherland et al., and Hirschfeld, there also exists the extra shortcoming that all intercalant dyes are known or suspected mutagens. Therefore, the troublesome issues of collection and disposal of hazardous chemical waste exists subsequent to each analysis.
  • the device may function without the need for external reagent treatment and obviated the need to collect and dispose of hazardous waste.
  • Such a technology then readily lends itself to automated and in-line analysis and precludes the need for skilled technicians to partake in the analysis procedure or disposal of waste (provided the sample itself is not biohazardous).
  • the other advantage provided by the incorporated dye is internal calibration. More specifically, three key advantages may be realized: 1) the associated dye provides a means to determine the quantity of fluorophore and immobilized nucleic acid on the waveguide; 2) the fluorophore in the presence of single-stranded nucleic acid provides a baseline signal to which all signals can be referenced, hence providing meaningful analytical data; and 3) the useful lifetime of the device can be determined from alterations in the background fluorescence signal from the incorporated fluorophore over time. Therefore, by including the associated fluorescence transduction unit, an internal reference marker and diagnostic tool for the device status is included as an integral part of the optical biosensor.
  • Nucleic acid oligomers are covalently immobilized onto optical fibers by first activating the surface of the optical fiber with a long chain spacer arm terminated by a chemically protected terminus, normally a dimethoxytrityl (DMT) moiety, followed by automated solid-phase DNA synthesis. Detection of nucleic acids or nucleic acid analogs at the fiber surface after hybridization between immobilized nucleic acid and its complementary nucleic acid is achieved by measuring enhanced fluorescence emission of the fluorophore.
  • the optical fiber may be activated with a number of different compounds.
  • the method of Arnold and co-workers may be used for the activation of the fused silica wafers, optical waveguides, and optical fibers whereby 25 atom-long spacer molecules terminated by a dimethoxytrityl protected nucleoside are immobilized onto the cleaned optical fiber substrate, as illustrated in Fig. 1 (a).
  • the length of the spacer between the substrate and the first nucleoside is sufficiently long so that the environment of the terminal nucleoside is fluid enough to permit efficient coupling with successive nucleotide monomers during automated phosphoramidite synthesis of the immobilized nucleic acid probe.
  • linker on the support is easily determined by determining the amount of trityl cation released during the first trichloroacetic (TCA) deprotection step of the automated synthesis.
  • TCA trichloroacetic
  • An amine-terminated solid support suitable for automated oligonucleotide synthesis may be prepared according to the method of Brennan et al. (1993, Sensors and Actuators B, VV. 109).
  • a bifunctional amphiphilic support derivatization agent is created by condensing ⁇ -aminopropyltriethoxysilane (APTES) with 12-nitrododecanoic acid.
  • APTES ⁇ -aminopropyltriethoxysilane
  • the resulting long chain spacer molecule is covalently immobilized onto the surface of the optical fibers by an S n 2 reaction between the hydroxyl groups present at the surface of the fiber and the silane moiety of the amphiphile.
  • the support may then be capped using standard methods employed during automated synthesis (acetic anhydride), or with chlorotrimethylsilane (R.T. Pon Methods in Molecular Biology. Vol.20: Protocols for Oligonucleotides and Analogs, S. Agrawa, Ed, 1993, Humana Press, Inc. Totowa NJ.), thereby masking other sites of reaction which may produce unwanted side products during oligonucleotide synthesis. Reduction of the terminal nitro-functionalities is then achieved by treatment of the derivatized support with an acidic zinc solution.
  • the resulting amine headgroups may then be used directly for automated synthesis wherein an ammonolysis/base resistant phosphoramidate linkage is made between the activated support and the first nucleotide.
  • An outline of a synthetic procedure used to immobilize alkyl amine monolayers covalently onto fused silica substrates is depicted in Figure 1(b).
  • the hydrolysis resistant linkage of Maskos and Southern may also be employed to provide waveguides functionalized with substrate linkers.
  • a phosphodiester linkage between the substrate linker and first nucleotide is completely resistant to ammonolysis under the conditions which remove standard base-protecting groups.
  • This linkage is produced by derivatization of optical fibers with the bifunctional silylating reagent 3-glycidoxypropyltrimethoxy silane via silyl-ether bond formation with the hydroxylated waveguide surface. This yields a substrate derivatized with short spacer molecules with terminal epoxide moieties.
  • the length of the spacer arm is then extended by nucleophilic attack of a polyether, such as hexaethylene glycol (HEG), in an acid catalyzed expoxide ring-opening reaction, yielding a stable ether linkage (U. Maskos and E.M. Southern, 1992 Nucl. Acids Res., 20(7). 1679), as shown in Fig. 1(c).
  • a polyether such as hexaethylene glycol (HEG)
  • HEG hexaethylene glycol
  • polyethylene glycols are bifunctional, there exists the possibility of creating non-reactive closed-loop structures which may significantly decrease the amount of loading of oligonucleotides on the surface of an optical fiber, as shown in figure 1(d).
  • a suitable blocking group for example, with a DMT functionality, prior to extension of the glycidoxypropyltrimethyl silane.
  • a chromophoric protecting group such as DMT
  • an additional advantage is provided wherein facile determination of the amount of support linkers may be determined by monitoring the absorbance of the deprotection solution (e.g. 504 nm for DMT).
  • Mono-dimethoxytrityl protected polyethylene glycols may be introduced onto the surface of fused silica waveguides by a number of methods. Waveguides first functionalized with GOPS, as in the method of Maskos and Southern, may then be treated with a solution of mono-dimethoxytritylated polyethylene glycol over sodium hydride to afford linkage of the polyether to the terminal epoxide moiety of the immobilized GOPS via a base catalyzed epoxide ring-opening reaction as shown in figure 1(e).
  • Mono-dimethoxytritylated polyethylene glycols can also be directly linked to the surface of fused silica waveguides by activation of the terminal hydroxyl moiety of the polyether with methane sulfonyl chloride or ⁇ - cyanoethyl N,N-diisopropyl phosphityl chloride, as shown in Figs. 1(e) and 1(f), respectively.
  • the polyether substrate linker is attached as a phosphoramidite synthon which can be done as part of the automated oligonucleotide synthesis procedure; thereby making the entire biosensor fabrication protocol completely automated after cleaned waveguide pieces are introduced into the synthesis column of the automated synthesizer.
  • the biorecognition element to be bound onto the terminus of the substrate linker in configuration of the described biosensor can include immobilized nucleic acids (DNA and RNA), modified nucleic acids, and nucleic acid analogs prepared by well-known methods or by straight-forward extension or modification of those methods.
  • nucleic acid includes polynucleotides, oligomers, relatively short polynucleotides (up to about 50 bases), longer polynucleotides ranging up to several hundred bases, and doubled-stranded polynucleotides.
  • nucleic acid analogs includes modified nucleic acids.
  • nucleotide analog includes nucleic acids where the internucleotide phosphodiester bond of DNA or RNA is modified to enhance bio-stability of the oligomer and "tune" the selectivity/specificity for target molecules (Ulhmann, et al, 1990, Angew. Chem. Int. Ed. Eng., 90: 543; Goodchild, 1990, J. Bioconjugate Chem., L 165; Englisch et al, 1991 , Angew, Chem. Int. Ed. Eng., 30: 613).
  • nucleic acid sequences are covalently attached to the surface of the optical fiber.
  • an automated DNA synthesizer is used to grow nucleotide oligomers onto the surface of activated optical fibers via the well established ⁇ -cyanoethylphosphoramidite method. Any commercially available automated DNA synthesizer can be used.
  • the use of an automated synthesizer to grow nucleic acids or nucleic acid analogs on the optical fiber substrates provides many advantages over conventional techniques of DNA immobilization.
  • nucleic acid strands are adsorbed onto a suitable support (usually nitrocellulose) with little known about strand orientation.
  • a suitable support usually nitrocellulose
  • the use of an automated oligonucleotide synthesizer provides full control of the oligomer sequence, strand orientation, and packing density in association with activation of the optical fiber substrates. Control over these parameters is critical to the development of a nucleic acid detection method based on hybridization as the alignment of the immobilized strands with respect to the availability of target nucleotides for hybridization and intermolecular interactions (electrostatic and steric) between oligomers will have direct ramifications on the kinetics and thermodynamics of hybrid formation and dissociation.
  • the use of a gene machine in addition to the chemistry used to activate the surface of the optical fibers, allows for the creation of membranes of desired density and structural order to permit rapid and reversible hybridization, and to control refractive index.
  • the method utilizing ⁇ -cyanoethyl phosphoramidites is preferable as complete deprotection of the oligonucleotides can be done using aqueous ammonia (as opposed to thiophenol) for the case where oligonucleotides were grown onto controlled pore glass (CPG).
  • Triethylamine is used to deprotect the ⁇ -cyanoethyl protected oligonucleotides grown onto fused silica wafers or optical fibers without liberating the oligonucleotides from the support.
  • An overview of the ⁇ -cyanoethylphosphoramidite synthesis is as follows:
  • the first step in each cycle of solid phase automated phosphoramidite synthesis involves the removal of the dimethoxytrityl protecting group on the immobilized nucleotide.
  • Detritylation is done by introducing a solution of 3% trichloroacetic acid (TCA) in 1 ,2 dichloroethane (DCE) onto the synthesis column in order to yield a 5'-hydroxyl functionality onto which the next nucleotide monomer may be coupled.
  • TCA 3% trichloroacetic acid
  • DCE dichloroethane
  • TCA is the reagent of choice for detritylation due to its rapid reaction rate so that the oligonucleotide is only exposed to the acid for short periods of time, thereby avoiding the acid catalyzed removal of the adenine and guanine moieties from the nucleotide sugar groups by the process of depurination.
  • the acid is removed by flushing the column with acetonitrile.
  • the eluent containing the released trityl cation is sent to a fraction collector so that the coupling efficiency of the synthesis may be monitored by absorption spectroscopy. Coupling is the next stage of the synthesis cycle.
  • the contents of the synthesis column are dried by alternatively washing with acetonitrile and flushing with dry argon. This ensures that the support is anhydrous and free of nucleophiles.
  • the next step of the synthesis is the capping step. This is done to eliminate further growth of sequences onto which coupling did not occur.
  • the failed sequences are rendered unreactive by introducing acetic anhydride in the presence of dimethylaminopyridine in order to acetylate any remaining unprotected 5'-hydroxyl moieties.
  • a solution of aqueous iodine is added after flushing the capping reagents from the column. This is done in order to oxidize the trivalent internucleotide phosphite moieties to the more stable pentavalent phosphate moieties found in naturally occurring nucleic acids. This procedure is termed the oxidation step. Following the oxidation step, one cycle of nucleotide addition is complete.
  • the process may be repeated many times until oligonucleotides of desired length and base sequence have been constructed. After addition of the last nucleotide, a final detritylation step is usually done in order to yield a ⁇ '-hydroxyl group on the completed sequence.
  • Triethylamine is used for the removal of ⁇ -cyanoethyl protecting groups on the internucleotidic phosphotriester moieties of oligonucleotides grown onto optical substrates. This procedure is known to cause quantitative loss of the phosphate protecting groups via a ⁇ -elimination mechanism while not cleaving the single-stranded nucleic acids from the optical fibers. Ammonia treatment of the immobilized oligonucleotides is avoided by choosing an all-thymine base sequence. Thymine does not contain a primary amine functionality which would require protection during oligonucleotide synthesis.
  • the phenoxyacetyl (PAC) family of protecting groups represents a convenient method for blocking the exocyclic amino functions of guanine, adenine and cytosine residues (thymine or uracil require no nucleobase protection).
  • the half-time of deprotection with concentrated ammonium hydroxide at 20°C is 8 min, 7 min and 2 min, respectively (Wu et al, 1989).
  • the cyanoethyl phosphate protecting groups are removed within seconds (Letsinger and Ogilvie, 1969), whereas the linkage which joins the oligomer to the surface of the fused silica fiber (e.g., a phosphodiester or phosphoramidate) is completely stable under these conditions.
  • Alternative labile protecting groups are derivatized phenoxyacetyl groups including alkyl substituted PAC groups, more specifically t- butylphenoxyacetyl groups.
  • the t-butylphenoxyacetyl group can be quickly removed compared to hydrolysis of the linkage to the spacer thereby reducing the possibility of cleavage of the immobilized sequence from the surface.
  • N- phenoxyacetyl deoxynucleoside 3'-cyanoethylphosphoramidites and the analogous t-butylphenoxyacetyl phosphoramidites are commercially available. It has been reported by Polushin and Cohen (N. N. Polushin and J. S.
  • labile protecting groups could include the "FOD" (fast oligonucleotide deprotection available from Applied Biosystems Inc.) based on N,N-dialkylformamidines (Vinayak et al, 1992, Nucleic Acids Research, 20: 1265- 1269). Kuijpers et al (Tetrahedron Lett., 1990, 3 . 6729-6732 and Nucleic Acids Res., 1993, 21, 3493-3500) have described a method of nucleobase protection using 2-(acetoxy-methyl) benzoyl (AMB) moieties which can be removed by treatment with anhydrous potassium carbonate in methanol for 90 minutes at room temperature.
  • AMB 2-(acetoxy-methyl) benzoyl
  • Free short strands of nucleic acids can also be covalently attached to the optical fiber directly or via linker molecules. This approach allows the use of DNA or RNA isolated from natural sources, amplified nucleic acids or their analogs, or synthetic samples provided in the fully deprotected form. Protocols provide end- attached oligomers of a well defined orientation. Chemically stable linkages between the support and oligonucleotide may be employed to enhance the robustness of the biosensor.
  • Quartz (or interchangeably fused silica) optical fibers derivatized with linker molecules terminated with either hydroxyl or amino groups can serve as substrates for carbodiimide-mediated coupling with terminally phosphorylated single-stranded nucleic acids. Coupling to the hydroxyl fiber produces a phosphodiester bond while coupling to an amine fiber yields a phosphoramidate bond. Oligonucleotides can be phosphorylated, in solution, either chemically via a modification of Ouchi's method (Sowa et al Bull. Chem. Soc, Japan 1975, 48 2084) or enzymatically.
  • Covalent attachment of free short strands of single-stranded nucleic acid to the optical fibers can be achieved by a slight modification of the method Ghosh and Musso (Ghosh and Musso, 1987, Nucl. Acids Res. 15: 5353). Coupling of a 5'-aminohexyl derivatized oligomer with activated carboxyl fibers affords end- attached oligomers. This method is known to minimize reaction at the amino groups of the DNA bases (which would potentially compromise the hybridization event) and affords surfaces with excellent nucleic acid coverage. The synthesis of the 5'- or 3'-terminally modified oligomers can be achieved readily by standard methods (Ghosh and Musso, 1987; Beaucage and Iyer, 1993).
  • RNA may be assembled on the support or prepared separately and linked to the support post-synthesis.
  • RNA monomers are commercially available, as are some 2'-O-modified synthons.
  • the 2'-O-methyl, allyl and 2'-deoxy-2'-fluoro RNA analogs, when incorporated into an oligomer show increased biostability and stabilization of the RNA/DNA duplex (Lesnik et al., 1993, Biochemistry, 32: 7832).
  • nucleic acid analogs also include alpha anomers ( ⁇ -DNA), L-DNA (mirror image DNA), 2'-5' linked RNA, branched DNA/RNA or chimeras of natural DNA or RNA and the above-modified nucleic acids.
  • Back-bone replaced nucleic acid analogs can also be adapted to use in the biosensor of the present invention.
  • PNAs peptide nucleic acids
  • PNAs peptide nucleic acids
  • carbamate-bridged morpholino-type oligonucleotide analogs Burger, D.R., 1993, J. Clinical Immunoassay, 16: 224; Uhlmann, et al., 1993, Methods in Molecular Biology, 20,. "Protocols for Oligonucleotides and Analogs," ed. Sudhir Agarwal, Humana Press, NJ, U.S.A., pp.
  • nucleic acid analogs are also embraced by the term “nucleic acid analog.” Both exhibit sequence-specific binding to DNA with the resulting duplexes being more thermally stable than the natural DNA/DNA duplex.
  • Other back-bone replaced nucleic acids are well-known to those skilled in the art and may also be used in the present invention (See e.g., Uhlmann et al 1993, Methods in Molecular Biology, 20, “Protocols for Oligonucleotides and Analogs," ed. Sudhir Agrawal, Humana Press, NJ, U.S.A., pp. 335).
  • Optical substrates such as planar wafers and optical fibers may be used in the present invention.
  • a preferred embodiment utilizes optical fibers.
  • Optical fibers are particularly advantageous as membrane supports due to their small size, high light transmission capability, and ability to allow total internal reflection (TIR) of light.
  • Optical fibers also provide a compact an rugged sensing device, and offer the ability to do remote spectroscopic measurements (Love et al, 1991 , Biosensors with Fiberoptics, D.L. Wise and L.B. Wingard (Eds.), Humana, NJ, pp. 139-180).
  • TIR total internal reflection
  • Extrinsic mode configurations are those in which the waveguide is simply used as a light pipe or conduit. End-on extrinsic mode investigations are usually done using optical fibers. In a biosensor which uses end-on extrinsic mode configurations, the fluorescent dyes and selective chemistry are located on or near the distal end of the fiber. The fiber is used as a light-pipe or conduit, where the excitation or emission radiation is simply guided from the sampling region to the detector. Fluorescence is stimulated by coupling excitation radiation into the near end of a fiber, and emission can be monitored by placing light sensing equipment directly opposite the distal end of the fiber.
  • the detector is placed at the near end of the fiber as some of the fluorescence may be coupled back into the fiber and totally internally reflected back to the near end.
  • the side-on extrinsic mode approach is typically used for investigations carried out on planar supports, but may also be used for fibers.
  • the immobilized single-strand nucleic acid and fluorophore are placed along the length of the optical fiber waveguide/wafer.
  • the fiber is illuminated by a light source located normal to the length of the fiber and fluorescence emission is also monitored by equipment placed normal to the fiber. Extrinsic configurations provide the advantage that simple and inexpensive equipment, including conventional light sources and detectors, are used (Krull et al, 1991 , Fiber Optic Chemical Sensors and Biosensors, Vol.
  • an intrinsic mode arrangement based on careful control of refractive index is used to monitor fluorescence emission from the surface of optical fibers.
  • Fluorophores present at either the surface or just below the surface of the fiber may be excited through the formation of a standing wave electric field which propagates normal to the surface of the fiber upon total internal reflection of radiation in the fiber.
  • the process of TIR occurs when the angle of incidence, ⁇ , at the interface between a fiber of high refractive index, n , and the external medium of lower refractive index, ⁇ 2 , is larger than a critical angle, ⁇ c , defined as:
  • the amplitude of the electric field of the reflecting radiation decreases exponentially as a standing wave into the medium having the lower refractive index.
  • This decaying radiation is referred to as an evanescent wave and can be used to excite fluorophores located near the boundary for TIR.
  • the propagation intensity, /, of the evanescent wave depends on the reflection angle, ⁇ , the wavelength of the transmitted radiation, ⁇ , and a Fresnel transmission factor, T:
  • the penetration depth is defined as the distance at which the intensity of the evanescent field has decayed to 1/e of the intensity at the reflection boundary.
  • the evanescent wave propagates into a thin zone beyond the surface of a fiber with a penetration depth ranging from about 200 nm to 400 nm for visible light.
  • Fluorophores within the evanescent wave propagation zone are excited by that evanescent wave to emit fluorescence. Fluorophores further from the interface with the optical fiber will experience lower intensity of light at the excitation frequency and a resultant concomitant decrease in intensity of emitted fluorescence.
  • a major limitation of the evanescent wave excitation is that less than 0.01 % of all of the excitation radiation on a fiber actually leaks beyond the fiber as an evanescent wave, and less that 2% of the fluorescence caused by the evanescent wave is actually recovered back into the fiber for transmission to the detector by total internal reflection (Love et al, 1991 , Biosensors with Fiberoptics, D.L. Wise and LB. Wingard (Eds.), Humana, NJ, pp. 139-180).
  • the evanescent wave mode of excitation and fluorescence signal recovery is very inefficient and not the preferred mode of operation for optical sensor devices.
  • the boundary for TIR effectively becomes the interface between the immobilized layer and the solution.
  • Fluorophores bound to nucleic acid in the immobilized layer are directly exposed to the electromagnetic radiation bound within the waveguide thereby providing a vastly improved excitation efficiency and, as a consequence, emit increased intensity fluorescence.
  • Fluorophores in the immobilized layer then emit fluorescence radiation within the waveguide itself to provide a much improved probability for transmission of the fluorescence signal by total internal reflection to the detector, yeilding increased sensitivity and lower target nucleic acid detection limits.
  • Fluorescence is the analytical method chosen for the transduction of hybridization events into a measurable analytical signal, since fluorescence techniques have long been known to provide high sensitivity (comparable to radioisotopic methods) and detailed information about structure at the molecular level (Lakowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press, NY). Changes in the polarity, pH, temperature, microviscosity, or orientation of molecules in the local environment of a fluorophore may result in alteration of the electronic structure or collisional probabilities of the fluorophore. Such environmental changes may be detected by monitoring fluorescent signal parameters such as intensity, wavelength, lifetime, or polarization.
  • the present invention utilizes, and is not limited to, the fluorescence intensity response of the bound fluorophore via monitoring in a total internal reflection configuration along the optical fiber substrate to quantify the presence of hybridized nucleic acids at the surface of the fiber.
  • the fluorescence intensity is directly proportional to the amount of target nucleic acid or nucleic acid analog initially present in solution. It is also possible to use the time dependence of the rate of change of the fluorescence intensity increase upon hybridization to determine the concentration of target nucleic acid.
  • the fluorophore of the present invention can be for example ethidium bromide (EB).
  • EB ethidium bromide
  • the ethidium cation (3,8-diamino-6-phenyl-5-ethyl- phenanthridium) is a fluorescent compound which strongly associates with double stranded nucleic acids by intercalation into the base-stacking region and, in some cases, the major groove of the double helical structure (Monaco et al., 1993, Journal of Bimolecular Structure and Dynamics, 10: 675).
  • the ethidium cation is particularly well suited for investigations of nucleic acid hybridization for a number of reasons.
  • the quantum yield of the dye is known to increase as much as 100-fold when intercalated into the base stacking region with respect to that of the unbound dye in aqueous solution (Bauer et al, 1989, Proceedings of the National Academy of Science USA, 56: 7937).
  • the binding affinity and the fluorescence enhancement of the dye are independent of base composition (Cuniberti et al, 1990, Biophysical Chemistry, 38: 11).
  • intercalation of the ethidium cation is known to increase duplex stability as the two 3,8-amino substituents hydrogen bond with the internucleotide phosphate groups on each of the DNA strands (whereas other intercalators are known to significantly decrease duplex stability) (Cuniberti et al, 1990, Biophysical Chemistry, 38: 11).
  • the absorption maximum of ethidium bromide is 510 nm, which is sufficiently close to the output wavelength of 488 nm of an Ar + laser which may be used to excite the fluorophore.
  • the dye has an emission maximum of 595 nm when bound to DNA which is a sufficiently large Stoke's shift to make separation of the emission radiation from the excitation radiation straight forward, and to prevent inner filter effects, by the use of a dichroic mirror or other standard optical components (Haugland, 1992, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, 5th Ed:, USA: Molecular Probes Inc.). Due to the above mentioned reasons, the use of EB has been shown to provide a sensitive means to detect the presence of nucleic acid duplexes for this application.
  • FIG. 3a A specific example of a tethered fluorophore is illustrated in the synthetic schemes of Figure 3a,b, and c.
  • a modified ethidium-type dye with tether here C 13 acid moiety
  • the ethidium analogue with acid tether is attached to 5'-hexylamine functionalized oligonucleotides immobilized on the surface of an optical fiber to generate the biosensor with the tethered fluorophore probe.
  • the 5'-hexylamine functionalization can readily be achieved through the use of the commercially available reagent Aminolink 2 ® .
  • the fluorophore or reporter group may be attached to the 5'- or 3'-end of the oligomer by not only a hydrocarbon tether but other types of tethers such as polyether, mixed aliphatic/aromatic, or peptidic.
  • the tether need not be restricted to the 3'- or 5'-ends of the oligomer but may be attached to a terminal or internal ribo-residue via the 2'-hydroxyl (Yamana et al, 1991 , Tetrahedron Letters, 32: 6347).
  • a tether can be attached to a terminal or internal nucleobase using pyrimidines (Pieles er al, I990, Nucleic Acids Research, 18: 4355) or purines (Roduit et al, 1987, Nucleosides and Nucleotides, 6: 349).
  • the internucleotidic linkage can be a site for tether attachment (Agrawal et al, 1990, Nucleic Acids Research, 18: 5419). Obviously, any combination of these methods could be used to site specifically incorporate multiple reporter groups.
  • Inorganic coordination complexes such as the "molecular light switch" Ru(phen')2 dppz PF ⁇ developed by Jenkins et al. (1992, J. Amer. Chem. Soc.
  • 114: 8736 may also be used as well as groove binding dyes, such as Hoechst 33258 and Hoechst 33342, which are commercially available from Aldrich Chemical Co. (Milwaukee, WI). These fluorophores are chosen such that the fluorescent probe is quenched (non- emissive) when in the presence of single-stranded nucleic acids and provides intense luminescence when in the presence of double stranded nucleic acids. This change in observed luminescence occurs via changes in the relative rates of radiative and non-radiative relaxation processes of the probe when the external environment changes from aqueous solution to a hydrophobic and highly structured one in the base stacking region of double-stranded nucleic acids.
  • groove binding dyes such as Hoechst 33258 and Hoechst 33342, which are commercially available from Aldrich Chemical Co. (Milwaukee, WI).
  • classes of fluorophores which can be used in the present invention include acridine dyes, phenanthrides, phenazines, phenothiazines, quinolines, alfatoxin, polycyclic hydrocarbons, oxirane derivatives, actinomyces, anthracyclinones, thiaxanthenones, anthramycin, mitomycin, platinum complexes, poiyintercalators, norphilin-A, fluorenes and fluorenones, furocoumarins, benzodipyrones and monostral fast blue.
  • Preferred dyes are also those that provide large Stoke's shifts, can be excited at long wavelengths and have large differences in fluorescence lifetime, quantum efficiency, and/or wavelength of excitation and emission when in solution as compared to when bound to hybridized nucleic acids.
  • Light emitted from fluorophores (after direct excitation) at the surface of the fiber is preferentially coupled back into the fiber and can be monitored by a photomultiplier tube (PMT) or any other suitable light detection equipment.
  • PMT photomultiplier tube
  • Increasing the length of coated fiber results in a greater optical path length and better sensitivity (Krull et al, 1991 , Fiber Optic Chemical Sensors and Biosensors, Vol. II, 0. S. Wolfbeis, Ed. , CRC Press, Boca Raton, pp. 315).
  • Direct excitation of fluorophores in an immobilized layer extending from the biosensor results in improved signal to noise ratio as interferences from background fluorescence in the bulk environment are avoided.
  • Fig. 4(a) One instrument used for fluorescence intensity measurements is based on a fluorescence microscope as described elsewhere (Brennan et al, 1990, Anal, Chim. Acta., 237: 253) and shown in Fig. 4(a).
  • An instrument as shown in Fig 4(b) may also be used in which the output from a suitable light source, for example an argon ion laser, is directed into an optical fiber via a lens with a numerical aperture which is equal to or greater than the numerical aperture of the nucleic acid functionalized waveguide when in the hybridization buffer solution used for analyte detection.
  • a suitable light source for example an argon ion laser
  • the excitation radiation may be coupled into a delivery fiber via a twisted optical fiber waveguide assembly such that all modes carried by the first fiber into which the excitation radiation was first coupled would be delivered to the second fiber to provide optimal excitation of fluorophores associated with the biosensor.
  • the excitation radiation may be totally internally reflected along the length of the delivery fiber to a sensing fiber functionalized with immobilized oligonucleotide and fluorophore. Coupling of the radiation between fibers may be achieved by abutting the distal terminus of the delivery fiber to the proximal terminus of the sensing fiber in a suitable non-fluorescent fiber coupler.
  • the terminus of the coupler is preferentially designed as a compression-fit end which provides a solution-tight seal to prevent contaminants from diffusing into the fiber coupler and causing drift in the analytical signal.
  • the sensing fiber is situated within in a small volume, stop-flow, hybridization chamber made of a suitable inert material with good thermal conductivity (e.g. stainless steel or titanium).
  • the temperature of the hybridization may be controlled by use of a suitable thermoelectric housing to provide rapid thermostating to the desired temperature and computer control.
  • the temperature of the solutions in the hybridization cell may be accurately determined ( ⁇ 0.2 °C) by use of a glass encapsulated thermistor incorporated into the hybridization cell.
  • Solutions delivery to the hybridization cell and sensing fiber may be done by use of a computer controlled pump (e.g. peristaltic pump) where all solutions originate from a computer controlled autosampier. Fluorescence emission from fluorophores associated with immobilized nucleic acid complexes was totally internally reflected within the sensing fiber. The portion of the light coupled back into the delivery fiber was directed towards an interference filter with the appropriate bandpass window for the emission of the fluorophore used with the optical sensor. Fluorescence radiation traversing the interference filter then enters a photomultiplier tube to provide a quantitative measure of the fluorescence intensity.
  • the radiation source can be a frequency doubled laser, a semiconductor laser, bright lamp or LED. Coupling into the waveguide can be accomplished with fiber couplers, and the detector can be an avalanche diode rather than a PMT.
  • the biosensor operates as follows.
  • the optical fiber with attached fluorescently labeled single-stranded nucleic acid is placed in a flow through cell and immersed in hybridization buffer solution.
  • hybridization occurs followed by intercalation (or other suitable ligand binding motif) and enhanced fluorescence emission of the attached fluorescent probe, as illustrated in figure 5.
  • Fluorescence intensity is monitored in a total internal reflection configuration wherein the optical fiber and organic coating form a waveguide to provide excitation to the surface immobilized nucleic acid and fluorescent probe, as well as to collect fluorescence emission. By monitoring the fluorescence intensity from the fiber, a measure of the amount of target nucleic acid in solution can be determined.
  • Regeneration of the biosensor can be achieved by thermal methods such as by elevating the temperature within the flow-through hybridization cell or by chaotropic methods in which solutions of highly polarized salts alter the hydrogen bonding structure of the solution to affect denaturation of the hybridized complex. In either case, the complex stability in the system is reduced to the point where hybridization is not energetically favorable and the complement strands are dissociated from the covalently immobilized oligomers and may be flushed out of the flow cell. Regeneration methods as described herein can be employed to recycle biosensors. Formation of multi-stranded nucleic acids (i.e. nucleic acid complexes composed of 3 or more strands), such as triplex nucleic acids, may be determined from the temperature dependence of the fluorescent signal.
  • the fluorescence efficiency of a fluorophore increases with decreasing temperature owing to the reduced collisional deactivation as a consequence of the reduced kinetic energy of the molecules surrounding the fluorophore. Fluorescence efficiencies with negative temperature coefficients are readily observed for fluorophores in solution and well as for fluorophores intercalated into nucleic acids, as illustrated in figure 6.
  • exclusion of the bound ligand often follows as the partition coefficient for the fluorophore in the multi-stranded nucleic acid is often much reduced with respect to that of the same fluorophore in double-stranded nucleic acid.
  • the ligand exclusion process will also show a temperature dependence where reduced ligand binding is observed as the temperature of the system is decreased.
  • a positive temperature coefficient of fluorescence intensity would be observed for fluorophores associated with multi-stranded nucleic acids as increasing amounts of fluorophore become excluded from the highly-structured environment within the nucleic acid complex into bulk solution where the probability for collisional quenching of fluorescence is far greater.
  • a net positive temperature coefficient of fluorescence intensity would then be observed for a fluorescent nucleic acid binding ligand in a multi-stranded nucleic acid.
  • the temperature at which multi-strand formation occurs could also be assayed from the maxima in a fluorescence intensity versus temperature plot where the temperature coefficient changes from negative (for the dye bound in double-stranded nucleic acid) to positive (for the dye being excluded from the multi-stranded nucleic acid complex). This process is illustrated in Figs. 7(a & b) for triplex formation on the sensor surface with linear and branched nucleic acids.
  • the biosensor of the present invention provides for rapid clinical testing for viruses (e.g., HIV, T cell lymphotropic virus 1 and 2, hepatitis B and C), and pathogenic bacteria (e.g. E. coli., Salmonella, Listeria, Chlamydia ssp., Trichomonas vaginalis, Gradenerelta vaginitis) as well as other microorganisms.
  • viruses e.g., HIV, T cell lymphotropic virus 1 and 2, hepatitis B and C
  • pathogenic bacteria e.g. E. coli., Salmonella, Listeria, Chlamydia ssp., Trichomonas vaginalis, Gradenerelta vaginitis
  • Detection of genetic disorders e.g., cystic fibrosis and sickle-cell anemia
  • diseases e.g. branched antisense nucleic acids which inhibit expression of targeted nucleic acid sequences via triplex formation with that particular sequence,
  • the biosensor is useful for the monitoring of the in-vivo response of bacteria to an antibiotic treatment to ensure the efficacy of the treatment regime.
  • the physician chooses both the antibiotic and the dosage to treat a bacterial infection. Samples of the infecting organism would be sent to the laboratory for MIC testing, Minimal Inhibitory Concentration for the lowest concentration of that particular antibiotic necessary to inhibit the growth of the infecting organism. If the MIC of the in-vitro test is less than the dosage given to the patient, the treatment is allowed to continue, otherwise an increased dose and/or a change of antibiotic would be ordered. The most significant problem of this approach is that of the time required, wherein 1 to 3 days are needed to acquire the MIC result.
  • the biosensor described above overcomes these problems by determining the concentration of one or more species of bacteria present in the patient. Samples would be taken at intervals and tested for the bacteria's concentration. The change in the bacterial concentration over time would reflect the efficacy of the antibiotic treatment against the infectious organism or organisms. This ensures that an adequate amount of an appropriate antibiotic would be provided to the patient without providing excessive amounts of the antibiotic.
  • the method described above may be used in a variety of situations to monitor response of organisms such as bacteria or fungi to drugs. Examples
  • Plastic-clad silica optical fibers with a diameter of 400 ⁇ m were purchased from 3M Specialty Optical Fiber (North York, ON, Canada). The cladding on the fibers was mechanically removed, and the fibers were cut to lengths of about 1 cm. One face on each fiber was polished by suspending the fiber over (and placing the end face of the fiber in contact with) the rotating plate of a Thermolyne type 37600 speed controlled mixer (Sybron Corporation, Dubuque, IO, USA) onto which 1200 grade emery paper was immobilized. All fused silica optical fibers were cleaned using a Harrick PDG-32G plasma cleaner (Harrick Scientific Corporation, Ossining, NY, USA) before activation with aminopropyltriethoxy silane (APTES).
  • APTES aminopropyltriethoxy silane
  • the fibers were then washed with a 1 :1 acetone/methanol mixture and stored in a vacuum desiccator.
  • the optical fibers were plasma cleaned for 5 minutes at low power (40 W) and were placed in a solution of 1 :200 (v/v) aminopropyltriethoxy silane (APTES) in dry toluene. This was done under a nitrogen atmosphere using glassware which was oven dried and previously treated with octadecyltrichlorosilane.
  • APTES aminopropyltriethoxy silane
  • the bis-succinate was reacted with N-hydroxysuccinimide and 5'-0-dimethoxytrityl-2'deoxythimidine in the presence of N.N'-dicyclohexyl-carbodiimide (DCC) and 4- dimethylaminopyridine (DMAP) to yield a nucleoside functionalized spacer molecule.
  • DCC N.N'-dicyclohexyl-carbodiimide
  • DMAP 4- dimethylaminopyridine
  • the surface is functionalized with spacer molecules of at least 25 A in length which had either an amine or a hydroxyl functionality at the terminus of the spacer molecule.
  • spacer molecules of at least 25 A in length which had either an amine or a hydroxyl functionality at the terminus of the spacer molecule.
  • a chemically resistant, non-hydrolyzable spacer molecule is employed. The method used was a modification of that reported by U. Maskos and E.M. Southern supra wherein the silica surface was treated with glycidoxypropyltrimethoxysilane (GOPS), followed by extension via treatment with hexaethylene glycol (HEG) under acidic conditions.
  • GOPS glycidoxypropyltrimethoxysilane
  • HOG hexaethylene glycol
  • the water soluble HEG linker will provide a more fluid environment (which should not self-assemble) so as to improve the ability of the immobilized DNA strands to hybridize with complementary material in solution (in terms of energetics and kinetics).
  • the hydrophilicity of the linker will also facilitate the removal of adsorbed contaminants (e.g. proteins, organics) which may occlude the surface and contribute to drift in the fluorescence intensity.
  • adsorbed contaminants e.g. proteins, organics
  • HEG is bifunctional, there exists the possibility of creating non-reactive closed-loop structures which may significantly decrease the loading of oligonucleotides on the surface of the fibers.
  • one terminus of the HEG is protected with a dimethoxytrityl functionality prior to extension with GOPS.
  • This strategy permits facile determination of the amount of support linkers bound to the silica surface. Removal of the trityl protecting groups by treatment with acid yields the highly colored trityl cation, which can be quantitatively measured by monitoring A (504 nm) of the deprotection solution. Knowing there is one trityl group released per linker molecule attached to the surface, the loading of HEG can easily be determined.
  • Immobilization of a protected linker molecule provides the additional advantage that the hydroxyl groups produced after the attachment of the HEG to the epoxide moiety and all other surface siianols can be capped to prevent unwanted oligonucleotide growth at these sites.
  • the additional charge imparted from the anionic backbone of a side product strand may inhibit hybridization between the analyte strands and neighboring probe sequences. See: R.T. Pon Methods in Molecular Biology, Vol.20: Protocols for Oligonucleotides and Analogs, S.
  • the buffer coating was mechanically stripped from the pre-cut optical fiber pieces (400 ⁇ m diameter x 44 mm) and the cladding was dissolved by treatment with acetone.
  • the fused silica substrates i.e., optical fibers or wafers, were added to a 1 :1 :5 (v/v) solution of 30% ammonium hydroxide/30% hydrogen peroxide/water and the mixture was stirred at 80°C for five minutes.
  • the substrates were then removed and treated with a solution of 1 :1 :5 (v/v) cone. HCI/30% hydrogen peroxide/water and the mixture stirred at 80°C for five minutes.
  • Dimethoxytrityl chloride (7.1g) was dissolved in 10 ml of dry pyridine and added dropwise to a stirred solution of hexaethylene glycol (5.65 ml) in 5 ml of dry pyridine under an argon atmosphere. Stirring was continued for 16 hours after which time the reaction mixture was combined with 50 ml of dichloromethane. The dichloromethane solution mixture was twice shaken with 900 ml portions of 5% aqueous sodium bicarbonate and then with three 900 ml portions of water in order to remove unreacted HEG, pyridine, and pyridinium salts.
  • the product was purified by liquid chromatography using silica gel and a solvent system of 0.1% triethylamine in 1 :1 dichloromethane / diethyl ether. The identity of the product was confirmed by proton NMR spectroscopy (200 MHz).
  • the cleaned fused silica substrates were suspended in a stirred solution composed of 40 ml xylene, 12 ml GOPS, and a trace of H ⁇ nig's base at 80°C overnight.
  • the fibers were then sequentially washed with methanol, chloroform, ether, and then dried in-vacuo.
  • GOPS functionalized fibers were suspended in a stirred solution of
  • the fused silica fibers functionalized with DMT-HEG were suspended in a solution of 1 :10 (v/v) chlorotrimethylsilane / pyridine for 16 hours under an argon atmosphere at room temperature.
  • DMT-HEG (0.5g) was suspended in 50 ml anhydrous pyridine. The solution was maintained under an anhydrous argon atmosphere and stirred, while 1.2 equivalents of methanesulfonyl chloride was added dropwise. The reaction allowed to proceed for 60 minutes at room temperature with stirring. The cleaned fused silica and silicon substrates were introduced into the solution containing the mesylated DMT-HEG and the substrate functionalization reaction allowed to proceed for 4 days with stirring at 40°C under an argon atmosphere.
  • DMT-HEG (0.5g) was suspended in a solution consisting of 12 ml of anhydrous THF and 4 ml of anhydrous N,N-diisopropyl ethylamine. The solution was maintained under an anhydrous argon atmosphere and stirred at all times. 1.1 equivalents of 2- cyanoethyl-N,N-diisoproylamino-phosphochloridite was added dropwise to the DMT-HEG solution and the reaction allowed to proceed for 90 minutes at room temperature.
  • the substrate linker functionalized optical fibers were placed into an emptied Applied Biosystems (ABI) Oligonucleotide Purification Cartridge column (OPC-column) or 10 ⁇ mol scale synthesis column with the dead volume being taken up by inert polyethylene pieces.
  • the end filter papers were replaced (ABI) and the column ends were crimped closed using aluminum seals.
  • Synthesis of oligomers onto the optical fibers was carried out at the 0.2 ⁇ mol. scale with a pulsed-delivery cycle in the "trityl off' mode.
  • the ⁇ - cyanoethylphosphoramidite cycle was used as supplied by ABI with the exception of extended nucleoside coupling times (2-10 min.) and increased solution delivery times to accommodate the larger synthesis columns.
  • a 30% ammonium hydroxide solution was drawn up into the synthesis column containing the optical fibers functionalized with immobilized oligonucleotides using a syringe and a male-to-male luer adapter.
  • the fibers submerged in ammonia were allowed to stand for two hours at room temperature after which time the ammonia solution was expelled from the column and the contents of the column were washed five times with 5 ml portions of sterile water.
  • the deprotection solutions and washings were collected and concentrated to a total volume of 1 ml. A 260nm of the concentrated deprotection solution was measured in order to determine the quantity of DNA liberated from the fused silica substrates.
  • the oligonucleotide sequence was (5'- TAG GTG AGA CAT ATC ACA GA-3 * ), which is a nucleic acid probe for the E03 forward sequence of the Candida albicans genome.
  • Fibers coated in ssDNA were either stored dry or kept in a solution of 1 :1 ethanol/water. All fibers were cleaned prior to use by sonication in a solution of 1 :1 ethanol/water for 5 minutes in order to remove any fluorescent contaminants adsorbed to the surface of the fibers.
  • oligonucleotide syntheses were evaluated by spectroscopic quantitation of trityl cation released during the trichloroacetic acid treatment steps of the automated synthesis.
  • the collected fractions of trityl cation were diluted with 2.0 ml of 5% TCA in 1 ,2-dichloroethane immediately prior to making absorbance measurements. Absorption at 504 nm was measured in order to determine the concentration and the total number of trityl cation moieties released during each TCA deprotection step of the synthesis. In this way, the total number of oligomers successfully grown onto the solid supports was determined.
  • Synthesis of dA 20 and rA 20 was done using a conventional LCAA-CPG support with the ⁇ -cyanoethylphosphoramidite cycle supplied by ABI.
  • a nonadecamer of random base composition (dR 19 ) was also prepared by simultaneously introducing all four phosphoramidite reagents to the column at each coupling step. Standard deprotection with aqueous ammonia (29%, 1.5 ml, 24 h) was used to liberate the oligomers from the solid support and remove the base protecting groups.
  • deprotection of the phosphate blocking groups, base protecting groups and cleavage from the CPG support was done by treating the oligomers with 1.5 ml of a solution consisting of 4 parts aqueous ammonia and 1 part ethanol for 48 hours at room temperature.
  • the aqueous solution containing the. oligonucleotides was then collected, evaporated to dryness, and the residue treated with 300 ⁇ l of an anhydrous solution of 1 M tetra-N-butyl ammonium fluoride in THF overnight at room temperature. After the incubation time, the reaction was quenched by adding 1 ml of water to the reaction mixture. Crude oligomer was purified by polyacrylamide gel electrophoresis and reverse phase liquid chromatography or size exclusion chromatography.
  • cDNA Complement DNA
  • cRNA Complement RNA
  • cRNA Non-complement Nucleic Acid by the Optical Sensor Fabricated by the Procedures of Examples 1 and 5.
  • An optical fiber functionalized with polythymidilic acid icosanucleotide was selected at random from the batch of fibers (ca. 25) created in example 1 and was positioned under the objective of the microscope, as illustrated in Fig. 4(a). In this orientation the incident laser radiation entered the proximal terminus and was totally internally reflected throughout the fiber. The majority of the fiber was submerged in a hybridization buffer solution consisting of 1.0 M NaCI and 50 mM sodium phosphate (pH 7.4) in sterile water contained within a 4 ml plastic cuvette.
  • Hybridization buffer was passed through an acrodisc ® filter immediately prior to introduction to the cuvette. Emitted fluorescent radiation, from the stimulated fluorescent molecules associated with the double-stranded nucleic acids, was directed back towards the microscope by total internal reflection. The emission from the fluorescent molecules was separated from the excitation radiation by a dichroic mirror and directed to a photomultiplier tube. The photomultiplier tube provided measurements of the intensity of fluorescence emission. Fluorescence intensity values are reported with the system at 25°C to avoid inconsistencies cause by the temperature dependence on fluorescence quantum efficiency and as relative quantities, thereby obviating the need to control experimental parameters such as laser intensity, optical alignment and photomultiplier tube (PMT) gain which are beyond accurate control from day to day.
  • PMT photomultiplier tube
  • dA 20 ssDNA was added to the plastic cuvette containing the suspended fiber in fresh hybridization buffer at a temperature of 85°C This temperature was chosen as it is sufficiently greater than the 60°C duplex melting temperature (T m , the temperature at which half of all the duplexes present are dissociated) and is well below the boiling point of the buffer. Incubation at temperatures below T m has been shown to cause incomplete hybridization wherein only a fraction of the bases on each strand interact to form partially hybridized complexes (Rubin et al, 1989, Nucleic Acid and Monoclonal Antibody Probes, B. Swaminathan and G.
  • the response of the fiber optic DNA biosensor to EB and cDNA is shown in Figure 9.
  • 10 ⁇ L of a 1 mg ml "1 aqueous solution of EB was added to the cuvette in which the fiber functionalized with ssDNA was suspended.
  • 60 ml of fresh hybridization buffer (25°C) was flushed through the cuvette in order to remove any non-specifically bound ethidium cation and no discernible increase in fluorescence intensity from the fiber was observed.
  • a 104 ⁇ 15% increase in the fluorescence intensity was observed from the fiber which was exposed to 189 ng ml "1 of cDNA and stained with EB relative to the baseline value for the cleaned sensor with only ssDNA on the waveguide surface.
  • the fiber was regenerated by flushing the cuvette and optical sensor 30 ml of hot (85 °C) buffer solution over a period of c.a. 30 seconds and the system allowed to stand for five minutes. After the five minute wait, an additional 30 ml of hot buffer was flushed through the cuvette to wash away the dissociated cDNA strands. This procedure is known to melt DNA duplexes as the buffer temperature was well above the T m of the dsDNA. The fluorescence intensity returned, within experimental uncertainty, to the initial intensity observed at the beginning of the experiment. The same hybridization experiment was repeated where the optical sensor was exposed to 757 ng ml "1 of cDNA during the hybridization procedure.
  • the staining time of the sensor with EB was changed after each hybridization with cDNA. For each determination, injections of 30 ⁇ l of 56.8 ⁇ g ml "1 solution of aqueous dA 20 were made and the hot hybridization buffer in the cuvette, which contained the cDNA strands, was allowed to cool to room temperature over a time of 30 minutes. A 1 mg ml "1 solution of EB in water (lO ⁇ l) was added to the cuvette after each hybridization to provide an EB concentration of 8.4 X 10 "3 M. A staining time of 20 min. with 8.4 X 10 "3 M EB was required to generate >99% of the full signal, as shown in Fig. 10(a).
  • the regression lines shown in figure 11 show good fits to the data points with r 2 values of 0.965 and 0.968 for the 1 month and 11 month-old fibers, respectively.
  • the sensitivity of the optical sensor (11 month-old, fabricated by the protocols of examples 1 and 5) was determined to be an increase in fluorescence intensity of 203% per 100 ng ml "1 of cDNA with a measured limit of detection of 86 ng-m . Maintenance of calibration has been observed for all experiments done thus far in which as many as 5 regenerations have been done over durations of up to 12 hours.
  • the samples were held at the low temperature limit for 15 minutes prior to initiating melt studies, to allow for thermal equilibration.
  • the temperature was then ramped at 0.5°C intervals at a rate of 0.5°C/min. while the absorbance was recorded at 260 nm.
  • Sequences of dT 20 were immobilized onto planar fused silica wafers (5 mm x 10mm x 1mm) according to the protocols in examples 1 and 5.
  • the immobilized dT 20 was hybridized with complementary dA 20 sequences by immersing the wafer in a 56.8 ng mr 1 solution of dA 20 at 85°C and allowing the immersed wafer to cool to room temperature (25°C).
  • the wafer was then removed from the cDNA solution and washed with room temperature hybridization buffer solution.
  • the wafer was then suspended in a quartz cuvette that was placed in the temperature controlled cuvette housing of the UV-vis spectrometer. The placement of the wafer was adjusted so that it rested in the path of the light beam.
  • the dead volume beneath the wafer was taken up by inert packing material.
  • Absorption spectra were collected at approximately 2°C increments of temperature in the range from 29°C to 76°C
  • the temperature in the cuvette was set by programming an external circulating bath to a specific temperature and the temperature of the buffer solution surrounding the fused silica wafer was quantitatively measured using a silanized glass encapsulated thermistor. Measurements of absorption at each temperature were done by integrating 100 spectra in the wavelength range between 220 nm and 320 nm.
  • the transition between an ordered duplex state and the disordered denatured state for systems of complementary nucleotides can be monitored and analyzed by UV- Visible absorbance spectroscopy to determine the duplex melting temperature (T m ).
  • T m duplex melting temperature
  • the extent of hybridization i.e. the number of base pairs formed per duplex was determined by a comparison of melt profiles for the immobilized oligonucleotides to similar reported values and dA 20 + dT 20 in solution.
  • fraction of single strands present in the system at any temperature (f ss (T)) may be determined through the use of the following equation:
  • A(T), A SS (T), and A ds (T) are the absorbances of the experimentally obtained melting curve, the upper baseline (single stranded oligomers), and the lower baseline (double stranded oligomers) respectively at temperature T (Nelson, J.W.; Martin, F.H.; Tinoco Jr., I. Biopolymers 1981, 20, 2509-2531.).
  • the purpose of the thermal denaturation studies was to examine whether linkage of an oligonucleotide to a solid support through a terminal nucleotide phosphate would cause the loss of degrees of freedom with respect to the availability of each nucleotide to partake in formation of the double stranded structure.
  • Melt profiles for the thermal denaturation of dsDNA immobilized on the surface of a fused silica wafer and dsDNA in solution were obtained, and the results of these investigations are summarized in Fig. 12.
  • the duplex melting temperature of the immobilized strands with aqueous phase complement strands was 62.4 ⁇ 0.3°C
  • the T m value for the aqueous phase dA 20 + dT 20 duplex was determined to be 60.5°C using the software supplied by Varian. Kibler-Herzog et. al. (Kibler-Herzog, L.; Zon, G.; Whittier, G.; Mizan, S.; Wilson, W.D. Anti-Cancer Drug Design 1993, 8, 65-79.) have reported the melting temperature of a dA 19 + dT 19 duplex in 1.02 M NaCI to be 61.1°C.
  • 12-hydroxydodecanoic acid (5g) was dissolved in 100 ml of dry methanol to which was added a solution of p-toluenesulfonic acid (88 mg) in 5 ml of methanol drop-wise over a 15 minute time-span. The solution was refluxed for 16 hours after which time the solvent was removed under reduced pressure. The product was then twice extracted into chloroform from a 5% aqueous solution of sodium bicarbonate. The organic phase was recovered, dried over NaS0 4 , and the solvent removed under reduced pressure.
  • 3,8-Dinitro-5-methyldodecanoate-6-phenyl-phenanthridium chloride (1.3mmol, 0.72g) was dissolved in 10 ml of THF and stirred over NiCI 2 *6H 2 0 (10.68g) and powdered Al (0.81 g). Water (0.3 ml) was then added to initiate the formation of the black Ni / Al catalyst and the reaction allowed to proceed for 15 minutes. The solution containing the reduced product was recovered by filtration, followed by removal of the solvent under reduced pressure. The product was purified by silica gel column chromatography (25% methanol in chloroform) and recovered as a dark purple solid (4%, 0.03g).
  • DMT-HEG-GOPS functionalized optical fibers prepared according to the method of Examples 2) were functionalized with polythymidilic acid icosanucleotide (according to the method of example 5) terminated an N- trifiuoroacetamide protected aminohexyl moiety at the 5'-end by use of the commercially available Aminolink 2 ® phosphoramidite synthon from ABI.
  • 5'-end of the immobilized strands was done by exposing the fibers to a 10 "3 M solution of sodium borohydride in absolute ethanol for 1 hour at room temperature. The fibers were then washed once in a solution of 10 "3 M HCI followed by washing with copious amounts of sterile water.
  • Fig. 14 The response of the reagentless sensor to 720 ng of complement DNA is shown in Fig. 14. Hybridization was done at 40°C in a buffer consisting of 1 M NaCI and 50 mM phosphate (pH 7.0). It should be noted that this sensor has a significantly improved response time over the sensors without tethered dye. 99% of the full analytical signal was reached in c.a. 6 minutes after injection of the complementary strands for the reagentless system while 45 minutes was required for full signal generation by the sensors without tethered dye (see figure 10 ⁇ a ⁇ ).
  • Binding modes can be characterized as: (i) intercalation of the ligand, in which typically a planar aromatic moiety slides between the DNA bases - stabilized by ⁇ - ⁇ stacking and dipole interactions, or (ii) minor or major groove interaction which is stabilized by hydrogen bonding, hydrophobic and/or electrostatic interactions (Long, E.C; Barton, J.K. Ace. Chem. Res. 1990, 23, 273.). Ethidium bromide binds to both duplexes and triplexes via an intercalative mode (Waring, M.J. Biochim. Biophys. Ada, 1966, 114, 234.), and this has been studied extensively by fluorescence methods.
  • the fluorescence quantum efficiency of the ethidium cation increases when intercalated into duplexes (LePecq, J.B.; Paoletti, C. J. Mol. Biol., 1967, 27, 87.) and triplexes (Mergny, J.L.; Collier, D.; Rougee, M.; Montenay-Garestier, T.; Helen, C. Nucleic Acids Research, 1991, 19, 1521., Scaria, PN.; Shafer, R.H. J. Biol. Chem., 1991, 266, 5417), however, it has been shown that there is a marked difference in the binding efficiency and hence fluorescence intensity between the two types of complexes.
  • LePecq and Paoletti were the first to observe that the fluorescence enhancement of ethidium during interaction with the duplex (poly rA) (poly rU) was significantly greater than for binding to the triplex (poly rA) (poly rU) 2 (LePecq, J.B.; Paoletti, C. C. R. Acad. Sc. Paris 1965, 260, 7033,).
  • triple-helical assay is an extension of work initiated for the detection of nucleic hybridization (Watson-Crick motif) using fiber optic total internal reflection fluorecence (TIRF) sensors functionalized with single-stranded deoxyribonucleic acid (ssDNA) probes.
  • TIRF fiber optic total internal reflection fluorecence
  • Optical fibers were activated by protocols given in example 2 and polyadenilic decanucleotides were assembled onto the substrate linker molecules on the fiber surface as per the methods given in example 5. Two batches of fiber were created, the first using commercially available N 6 -phenoxyacetyl-5'-0-DMT- 2'deoxyadenosine-3'-0-[( ⁇ -cyanoethyl)N,N-diisopropyl]phosphoramidite from
  • N 6 -phenoxyacetyl-3'-0-DMT-2'deoxyadenosine-5'-0-[( ⁇ - cyanoethyl)N,N-diisopropyl] phosphoramidite was prepared via standard protocols and used to grow oligonucleotide on the functionalized fibers in a reversed (fiber ⁇ substrate linker ⁇ 5'-dA10-3') orientation. Hi) Synthesis of Branched Oligonucleotides.
  • the "V" branched sequence 1 ( Figure 15) was synthesized on an Applied Biosystems 381A instrument using a 1 ⁇ mol scale synthesis cycle and ⁇ - cyanoethylphosphoramidite chemistry. Purification, desalting, and analysis of the branched oligonucleotide 1 by polyacrylamide gel electrophoresis was accomplished by our detailed protocols (Damha, M.J., Ganeshan, K., Hudson, R.H.E., Zabarylo, S.V., (1992) Nucleic Acids Res 20, 6565-6573; Damha, M.J.; Ogilvie, K.K.
  • Absorbance versus temperature profiles of the nucleic acid complexes were measured at 260 nm using a Varian Cary I UV-VIS spectrophotometer equipped with a variable temperature cell holder controlled by an external variable temperature circulating bath. Data were collected with the spectrophotometer set on dual beam optical mode to reduce optical drift. The data were collected at 260 nm at 0.5 °C intervals with an equilibration time of 60s for each measurement point. Absorption coefficients of the branched molecules were assumed to be similar to their corresponding linear sequences and were calculated from those of mononucleotides and dinucleotides according to the nearest-neighbor approximation (Puglisi, J.D.; Tinoco, I., Jr.
  • Samples for thermal denaturation analysis were prepared by mixing the pyrimidine containing strand with the target (2mM), lyophilizing the solution to dryness, and dissolving the oligomers in 10 mM Tris, 50 mM MgCI 2 , pH 7.3 adjusted with HCI. The mixtures were then transferred to Hellma QS-1.000-104 cells. Oligonucleotide solutions were heated to 80°C for 15 min and then slowly cooled to room temperature prior to melting experiments.
  • the laser radiation exiting the immersion lens of the fluorescence microscope (as described in Example 9) was coupled into a delivery fiber of similar numerical aperture (0.48) aligned beneath the objective, as illustrated in Fig. 4(c).
  • the light was totally internally reflected along the delivery fiber to a sensing fiber functionalized with immobilized oligonucleotide. Coupling of the radiation between fibers was achieved by abutting the distal terminus of the delivery fiber to the proximal terminus of the sensing fiber. A loss in optical transmission of no greater than 2% was observed for the coupled system.
  • the termini of the teflon fiber coupler were designed as compression-fit ends which provided a solution-tight seal that prevented contaminants from diffusing into the fiber coupler and causing drift in the analytical signal.
  • the sensing fiber was placed in a small volume, stop-flow, stainless steel hybridization chamber (1.5mm i.d. x 48mm) which provided a solution volume of 79 ⁇ l exposed to the sensing fiber.
  • the temperature of the hybridization cell was controlled by placing the cell in a thermostated housing in which glycol solutions from external variable temperature circulating baths were made to flow.
  • the temperature of the solutions in the hybridization cell were accurately determined ( ⁇ 0.2°C) by use of a glass encapsulated thermistor incorporated into the hybridization cell and in contact with the solution at the exit of the hybridization chamber. Solutions containing hybridization buffer, ethidium bromide, and complementary nucleic acid sequences were delivered to the hybridization cell and sensing fiber by use of a peristaltic pump.
  • Fluorescence emission from ethidium bromide that was intercalated into immobilized nucleic acid complexes was totally internally reflected within the sensing fiber. The portion of the light coupled back into the delivery fiber was directed towards the microscope objective where it was collimated and directed to the dichroic mirror. The fluorescence radiation was of longer wavelength ( ⁇ max 595 nm) than the dichroic cut-off, and was transmitted through the mirror and directed towards a photomultiplier tube, where the fluorescence intensity could be quantitatively measured. Drift caused by variations in the efficiency of optical coupling, laser intensity and photomultiplier gain were obviated by normalization of all signals to that of a standard solution of ethidium bromide at 25°C prior to and at the completion of each analysis.
  • the third dT 10 strand interacts by means of Hoogsteen hydrogen bonds with the dA 10 strand in target Watson-Crick duplex, and is oriented parallel to it.
  • the triplex 2 x dT 10 / dA 10 has two clearly resolved transitions, one for dissociation of the third strand from the duplex, i.e., dT 10 *dA 10 / dT 10 ⁇ dT 10 + dA 10 /dT 10 (T m 18°C), and one for dissociation of the duplex into its component strands, i.e., dA 10 / dT 10 ⁇ dA 10 + dT 10 (T m 32°C).
  • association of the third (dT 10 ) strand with the duplex (dA 10 / dT 10 ) is thermodynamicaily weaker than duplex formation itself.
  • Branched oligonucleotides are useful probes for stabilizing triplex DNA (R. Hudson, A. Uddin, and M. Damha J. Am. Chem. Soc, 1995, 117, 12470.).
  • the branched oligomer 1 (Fig. 15) for instance, binds to dA 10 to give a novel TAT triple-stranded complex in which both dT 10 strands are antiparallel to the purine (dA 10 ) strand.
  • this motif had been observed for TAT bases in complexes dominated by pur*pur/py bonding (e.g. G «GC, A «AT) ( ⁇ i ⁇ Moser, H.E.; Dervan, P.B. Science, 1987, 238, 645.
  • this triplex was induced by linkage of two dT 10 strands through their 5' ends via coupling to riboadenosine at the neighboring 2' and 3' oxygen atoms (Fig. 15).
  • This arrangement causes the initial direction of the two dT 10 strands to be parallel, and forces the formation of a triplex in which the third dT 10 strand runs antiparallel to the dA 10 strand, and is bound to it via reversed-Hoogsteen interactions.
  • Fig. 16(a) illustrates that as the dT 10 :dA 10 duplex was formed by lowering the temperature, there was an increase in the fluorescence intensity corresponding to ethidium bromide intercalation and quantum yield enhancement of the ligand in this complex.
  • the Hoogsteen triplex migrated more slowly than the duplex due to the presence of the third dT 10 strand.
  • the reversed-Hoogsteen triplex has the slowest mobility of all, and is characteristic of branched nucleic acid structures [ref Hudson and Damha, JACS 1993; Wallace, J.C.; Edmons, M. PNAS vol. 80, 950-954, 1983).
  • Association of 1 and dA 10 was quantitative as evidenced by the complete disappearance of compound 1 and dA 10 , when mixed in equimolar amounts.
  • the stoichiometry of interaction between dT 10 and dA 10 for the duplex and Hoogsteen triplex was also confirmed by studies at different concentrations of the two oligonucleotides.
  • Angularly dependent light scattering experiments were done to determine the refractive index of oligonucleotide monolayers covalently immobilized onto fused silica substrates.
  • the mode of operation of the devices namely, intrinsic or evanescent total internal reflection fluorescence
  • the concept of the experiments done is based on classical optical theory with respect to how alterations in the direction of a ray or collimated beam of light traversing an interface between two dielectric materials may be predicted based on the difference in the refractive index of the two materials, and vice versa.
  • n 1 sin(6' l ) n 2 sin(0 t ) (4)
  • Fig. 18(a) illustrates this concept for the case where the upper medium is fused silica and the lower medium is the ambient, as characterized by n Fused Sll ⁇ ca and n Ambl ⁇ nt , respectively, where t7 Fused Sll ⁇ ca > n Ambl ⁇ nt .
  • ⁇ c The angle of incidence for which this occurs is known as the critical angle, ⁇ c , and can be calculated from the following relation:
  • the incident ray undergoes total internal reflection (TIR) at the interface.
  • TIR total internal reflection
  • the angle of the reflected beam with respect to the normal to the interface is then equal to that of the angle of incidence for all 6
  • the critical angle for TIR can be determined directly from this point on the plot of scatter intensity versus incidence angle (Fig. 18(c)).
  • the refractive index of one of the media is known, the refractive index of the other can be solved using equation (5).
  • a three-layer model must be considered for the case where a thin film of organic material is placed at the interface, as shown in Figs. 19 and 20.
  • Each medium type is herein characterized by the refractive index of the material, as given by n Fused S ⁇ lca , n Fl m , and ⁇ Amb ⁇ ent , respectively, for the fused silica, organic film, and ambient.
  • the interaction of a light ray at each interface must be considered independently.
  • An incident ray in the fused silica medium at an angle ⁇ 9, relative to the interfacial normal will be refracted to a differing angle after traversing each interface. The propagation angle of the ray will then be ft and ft in the organic film and ambient media, respectively, relative to the interfacial normal.
  • the refractive index of the organic film can be directly determined from analysis of traces of scatter intensity versus ⁇ ,.
  • the critical angle for the TIR at the fused silica-film interface can be directly obtained from the point where the second local maxima intersects the baseline scatter intensity, as described previously for the two-layer model and shown in Fig 19(d).
  • n Fuse ⁇ Slllca , /7 Rlm and equation 4 Using the value of ⁇ , from the point where the first local maxima intersects the baseline scatter intensity, n Fuse ⁇ Slllca , /7 Rlm and equation 4, a second method for calculating 0 is provided. The goodness of agreement between the two values of ft would indicate the validity of the calculated value of n Fl ⁇ m .
  • n Ftlm For the case where t7 Fused Sl ca ⁇ n Fllm > t7 Amb ⁇ ent , an estimate of the value for n Ftlm may be attained provided the values of n Fused Sll ⁇ ca , t7 Amb ⁇ ent are known.
  • a direct determination of n m cannot be achieved in this case as TIR will not occur at the interface between the fused silica and organic film for light incident in the fused silica, yielding no mechanism for the determination of ft .
  • the ray diagrams and detector response trends for this scenario are shown in Fig. 20.
  • n F ⁇ m A slight underestimate for the value of n F ⁇ m may be had by assuming that # CF;ta/ (m4to ⁇ , is equal to the value of ⁇ , at the termination point of the local maxima from the plot of scatter intensity versus incidence angle.
  • This overestimate of ft and ⁇ Amb , ent can be used in equation 5 to provide an underestimate of the value of n Fllm .
  • This value of t7 Fllrn along with those for n Fused Slllca and ⁇ , can then be substituted into equation 4 to provide an underestimate of ft
  • This underestimate of ft and ⁇ Amb ⁇ ent can again be used in equation 5 to provide an overestimate of n F , ⁇ m .
  • An average of these two values should provide a good estimate of the true value of ⁇ F , ⁇ m to within the uncertainty limits set by the low and high value extremes.
  • Planar Suprasil ® fused silica wafers (Heraeus Amersil, Duluth, GA, USA) with dimensions of 10 x 5 x 1mm, a refractive index of 1.46008 and a surface flatness of 10 waves/inch, were functionalized with substrate linker molecules by the methods of examples 2 and 3.
  • silicon wafers (Heraeus Amersil, Duluth, GA, USA) with dimensions of 10 x 5 x 1mm were functionalized with substrate linker molecules by the methods of example 3.
  • Polythymidilic acid icosanucleotides were then assembled onto the functionalized wafers by automated solid-phase oligonucleotide synthesis, as per the methods provided in example 5.
  • the hybridization buffer was the same as that used for hybridization experiments on optical fibers and described in example 8.
  • the refractive index of the hybridization buffer was determined by use of an Bausch & Lomb Abbe-3L Refractometer (Fisher Scientific, Nepean, ON, CA) to within the reported accuracy of 0.0001.
  • Octadecyltrichlorosilane (OTS), ethylene glycol, hexadecane, carbon tetrachloride, chloroform and cyclohexane were of analytical grade or better from Aldrich Chemical Co. (St. Louis, MO, USA) and used as received unless stated otherwise.
  • Fused silica wafers were cleaned by treatment with solutions of NH 4 OH / H 2 0 / H 2 0 2 and HCI / H 2 0 / H 2 0 2 respectively, as per the method detailed in example 2 (i).
  • Prior to use carbontetrachloride and chloroform were dried by reflux over P 2 0 5 under an argon atmosphere followed by distillation under the same conditions.
  • Functionalization of the substrates with OTS monolayers was then done as per the methods of von Tscharner and McConnell (von Tscharner, V. and McConnell, H.M., Biophys. J, 36 (1981) 421) and as described in the following.
  • the cleaned substrates were treated with a solution of 80% hexadecane, 12% carbon tetrachloride, 8% chloroform and 0.1% OTS (v/v) for 15 minutes at 25°C with stirring under an anhydrous argon atmosphere.
  • the reaction mixture was then decanted and the functionalized fused silica wafers were then washed thrice with distilled chloroform and stored in-vacuo and over P 2 0 5 until required.
  • Wafers were placed in a custom-built stop-flow cell, beneath a Harrick EA 7X89 fused silica hemispherical prism with a radius of 8mm (Harrick Scientific Corp., Ossington, NY, USA), as illustrated in Fig. 21.
  • the other face of the wafer was exposed to a solution compartment with dimensions of 9 x 2 x 1 mm (I x w x h).
  • the flow cell was mounted at the vertex of a modified goniometer element obtained from a type 43702-200E Thin Film Ellipsometer (Rudolph Research Corporation, Flanders, NJ, USA) with an angular accuracy and precision of 0.005°.
  • 543 nm optical radiation from a Gre-NeTM Laser (Melles Griot, Carlsbad, CA, USA, 1mW output power, 1.5 mm beam diameter, 0.01 mrad beam divergence) mounted on one arm of the goniometer was passed through the hemispherical prism and impinged on the planar fused silica wafer.
  • the hemispherical prism guaranteed that alterations in the incidence angle owing to refraction at the air-prism interface were eliminated as the beam invariably entered the prism normal to the prism / air interface.
  • a M062-FC03 Slo-Syn stepping motor (Superior Electric Co., Bristol, CT, USA, 200 steps per revolution) was coupled via a set of gears to the screw shaft of the goniometer used to drive the pivoting mechanism of the goniometer arms.
  • the gear ratio used provided the motor with a 7 ⁇ mechanical advantage so as to reduce the load on the motor and prevent it from slipping.
  • TTL signals from a standard PC parallel interface were used to operate the advance mechanism of the stepper motor so as to offer accurate control of the angle of the goniometer arms and incidence of the laser beam.
  • One end of a fiber-optic bundle (Oriel Corp, Stratford, CT, USA, model no. 77533) was mounted in the base of the flow cell ca. 1 mm from the exposed face of the fused silica wafer.
  • the other terminus of the fiber bundle was directed to a 630nm long-pass colloidially-colored glass filter (Schott Glass Technologies, Duryea, PA, USA) placed before the window of an R-928 photomultiplier tube (Hamamatsu Corp., Bridgewater, N.J., USA) operated using a DZ-112 Photoelectric Indicator (Rudolph Research, Flanders, N.J., USA).
  • the long-pass filter provided for attenuation of the light intensity transmitted by the fiber bundle by a factor of 10 5 . This was done in order to prevent an overload condition in the PMT form occurring, guarantee the linearity of response and preserve the useful lifetime of the detector.
  • the current from the PMT was converted to an analog voltage output (0 - 5 VDC) from the signal processing electronics contained within the Photoelectric Indicator and passed to a 12 bit analog to digital converter (Metra-Byte, Taunton, MA, USA) for data acquisition on a PC computer using software created in-house to acquire plots of intensity versus incidence angle.
  • a monolayer film of OTS was covalently attached to the surface of a fused silica wafer by a method previously shown to provide dense surface packing and a theoretical refractive index in the range of 1.4 -1.6. ( ⁇ aJDucharme, D. et al., J. Phys. Chem, 94 (1990) 1925. ⁇ b ⁇ von Tscharner, V. and McConnell, H.M.; Biophys J., 36 (1981) 421). The results of the light scattering experiments are shown in Fig 23(c) and 23(d), respectively, for OTS functionalized fused silica wafers exposed to air and water as the ambient.
  • ellipsometry was done in order to provide secondary confirmation of the experimentally determined values of the refractive index for the oligonucleotide monolayers. Ellipsometry was done on samples of silicon wafer functionalized with substrate linker molecules by the methods of example 3 onto which molecules of polythymidilic acid icosanucleotide were assembled by automated solid-phase oligonucleotide synthesis as detailed in example 5. Silicon wafers were necessarily used as the substrate material for these experiments as the fused silica substrates used for the light scattering experiments provide little reflection of the laser beam incident at an angle 70° in the ambient.
  • the surface of the silicon wafers was made similar to that of fused silica via the cleaning procedure used prior to functionalization of the substrate.
  • This cleaning procedure is known to provided a layer of oxidized silicon at the surface of the silicon wafers (Kern, W. and Puotinen, D.A.; RCA Review, 31 (1970) 187-206).
  • silanol moieties then present at the oxidized silicon- ambient interface provide attachment points for the substrate linker molecules.
  • McCracken (F.L. McCracken, NBS Technical Note 479, Washington DC (1969)) has developed software capable of providing values of thickness and refractive index from ellipsometric measurements of thin films using the exact Drude equations for ellipsometry.
  • the Film 85 software provided with the AutoEL- II null reflection ellipsometer (Rudolph Research Corp., Flanders, NJ, USA) was based on that originally developed by McCracken and used for the analysis of ellipsometric data from the experiments described herein.
  • Ellipsometric analysis of the cleaned substrate revealed the formation of a 2 ⁇ A thick layer of oxidized silicon on the surface of the wafers. Three silicon wafers functionalized with substrate linker and oligonucleotide where then analyzed. Ten different locations on the wafer surfaces were chosen at random and the results of the ellipsometric analysis are summarized below in Table 3.
  • Maxwell-Garnet theory R.M.A. Azzam and N.M. Bashara, Ellipsometry and Polarized Light. North Holland Publishing Company, New York (1977), p. 359.
  • the concept of Maxwell-Garnet theory, as applied herein, is based on the notion that a partially formed monolayer film of coverage ⁇ is optically equivalent to a fully formed monolayer film of refractive index (n FHm ) and relative thickness (T f ) such that the observed film thickness (7) is related to that of the fully formed film by:
  • the same scaling factor, ⁇ can be applied to the refractive index value given from ellipsometric analysis so that values more representative of that for the actual immobilized layers can be obtained.
  • the results after applying this correction are given in Table 3 and provided an average value of 1.6 ⁇ 0.1 for

Abstract

L'invention concerne un biocapteur qui permet d'analyser directement l'hybridation des acides nucléiques au moyen d'une fibre optique fonctionnalisée avec des molécules d'acide nucléique et d'une transduction par fluorescence. Les sondes d'acide nucléique, immobilisées à la surface des fibres optiques, subissent une hybridation avec des acides nucléiques complémentaires introduits dans l'environnement local du capteur. Les événements d'hybridation sont détectés au moyen de composés fluorescents qui se lient de façon à former des hybrides d'acide nucléique. L'invention peut être utilisée pour détecter et cribler des troubles génétiques, des virus et des micro-organismes pathogènes. Ses applications en biotechnologie sont représentées par la surveillance des cultures et de l'expression des gènes, ainsi que du rendement (c'est-à-dire la dose-réponse) de produits pharmaceutiques destinés à la thérapie génique. L'invention concerne des systèmes biocapteurs dans lesquels des molécules fluorescentes sont liées aux molécules d'acide nucléique immobilisées. La méthode préférée pour immobiliser les acides nucléiques est la synthèse en phase solide in situ. L'indice de réfraction de l'acide nucléique immobilisé est déterminé par les solutions chimiques de dérivatisation du support et la synthèse des acides nucléiques. La dérivation des fibres optiques préférée crée un revêtement d'ADN ayant un indice de réfraction supérieur à l'âme de la fibre sur sa surface.
PCT/CA1998/000402 1997-06-18 1998-04-30 Diagnostic par biocapteurs a acides nucleiques WO1998058079A1 (fr)

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AU70244/98A AU755913B2 (en) 1997-06-18 1998-04-30 Nucleic acid biosensor diagnostics
JP50343899A JP2002511934A (ja) 1997-06-18 1998-04-30 核酸バイオセンサ診断装置
EP98916750A EP0991777A1 (fr) 1997-06-18 1998-04-30 Diagnostic par biocapteurs a acides nucleiques
US09/446,222 US6503711B1 (en) 1997-06-18 1998-04-30 Nucleic acid biosensor diagnostics
NZ502303A NZ502303A (en) 1997-06-18 1998-04-30 Nucleic acid biosensor system & diagnostic use
CA002294603A CA2294603A1 (fr) 1997-06-18 1998-04-30 Diagnostic par biocapteurs a acides nucleiques

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CA002208165A CA2208165A1 (fr) 1997-06-18 1997-06-18 Diagnostics grace a un biodetecteur d'acide nucleique
CA2,208,165 1997-06-18
US5097097P 1997-06-19 1997-06-19
US60/050,970 1997-06-19

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US10/338,787 Continuation US20030157538A1 (en) 1997-06-18 2003-01-07 Nucleic acid biosensor diagnostics

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US20030157538A1 (en) 2003-08-21
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US6503711B1 (en) 2003-01-07
AU755913B2 (en) 2003-01-02
EP0991777A1 (fr) 2000-04-12

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